miR-199-3p Suppresses Cellular Migration and Viability and Promotes Progesterone Production in Goose Ovarian Follicles Before Selection by Targeting ITGB8 and Regulating Other ECM-related Genes

ABSTRACTThe extracellular matrix (ECM) constitutes the follicular basal lamina and is also present between follicular cells. Remodeling of the ECM is believed to be a key event in follicular development, especially follicular selection, and plays important roles in cell migration, survival, and steroidogenesis. miR-199-3p is differentially expressed in the goose follicular granulosa layer during follicular selection and is reported to play a primary role in inhibiting cell migration and invasion. Nevertheless, the effect of miR-199-3p on ovarian follicles and its role in follicular cellular migration are not understood. In this study, we demonstrated by qRT-PCR that miR-199-3p was differentially expressed in the granulosa layer from goose ovarian follicles before and after follicular selection. Additionally, we found that miR-199-3p overexpression could significantly suppress cell viability and migration, as well as elevate both the concentration of progesterone and the expression of key progesterone production genes in cultured granulosa cells (GCs) from goose pre-hierarchical follicles. Furthermore, using dual-fluorescence reporter experiments on 293T cells, we confirmed that miR-199-3p downregulated the expression of the ECM gene ITGB8 by directly targeting its mRNA three prime untranslated region (3′ UTR). Finally, we found that miR-199-3p overexpression in the GCs of goose pre-hierarchical follicles inhibited the expression of two ECM-related genes (MMP9 and MMP15) yet promoted the expression of another two ECM-related genes (COL4A1 and LAMA1). Taken together, these findings suggest that miR-199-3p participates in granulosa cell migration, viability, and steroidogenesis in goose ovarian follicles before selection by targeting ITGB8 and modulating other ECM-related genes. These data highlight the key roles of miR-199-3p in follicular cell migration, viability, and steroidogenesis by regulating ECM-related genes and thus contribute to a better understanding of the mechanisms underlying follicle selection in birds.

Thermotolerance for Physiological and Endocrine Regulation of Embryo-Uterine Development

Hyperthermia affects most aspects of reproductive performance in mammals by compromising the physiology of reproductive tract, through hormonal imbalance, disrupting the development and maturation of oocyte, causing embryonic mortality, abortion, growth retardation, and major developmental defects. Heat stress reduces the steroidogenic capacity of its theca and granulosa cells by altering the efficiency of follicular selection resulting in drop of luteinizing hormone and estradiol secretions from the dominant follicle in the plasma, reduced intensity, and duration of estrus expression. The mechanism for the developmental stage-dependent change in heat tolerance is considered to be the accumulation of antioxidants in embryos in response to heat-inducible production of reactive oxygen species. Morula or blastocysts can repair heat-induced misfolded or unfolded proteins or facilitate DNA damage induced apoptosis. Therefore, embryo transfer (ET) that can bypass the heat-sensitive stage could be a good solution to improve the conception rate under heat stress. However, further research is required to improve the reduction in pregnancy rates due to summer heat stress.

Effects of stereotactic surgery on the anterior hypothalamus (HA) on the estrous cycle: Role of the dopaminergic system in spontaneous ovulation in the rat

We evaluated the function of dopaminergic receptors (DAR) of the anterior hypothalamus (AH) on the estral cycle (EC) regulation and spontaneous ovulation by a single microinjection (MI) with the dopaminergic antagonist haloperidol (HLP) in adult rats. One hundred thirty nine rats that exhibit forth-day estral cycles (cyclic animals: CA) received a stereotaxic surgery (STXS) on the right, left or both AH sides and were distributed in three different groups with a MI of 1 µL of: HLP (15 µg) or dimethyl-sulfoxide (vehicle) or other false MI group. All the animals with STXS were sacrificed in next vaginal estrus (VE) exhibited and the ova shed (OS) counted. In sixteen AC, the OS were counted at VE and forming a control group. The STXS affected the animals EC: just 59/139 exhibited a short EC (SEC) with 4.6±0.1 days compared with 80/139 that exhibited a long EC (LEC) of 13.6±0.2 days. False or HLP MI diminished OS just in animals exhibiting a SEC. STXS affects neuroendocrine processes controlling EC length when cutting dorsal connections to AH. The DAR of the AH participate on ovarian mechanisms of follicular selection.

Smad9 is a key player of follicular selection in goose via keeping the balance of LHR transcription

ABSTRACTThe egg production of poultry depends on follicular development and selection. However, the mechanism of selecting the priority of hierarchical follicles is completely unknown. Smad9 is one of the important transcription factors in BMP/Smads pathway and involved in goose follicular initiation. To explore its potential role in goose follicle hierarchy determination, we first blocked Smad9 expression using BMP typeⅠreceptor inhibitor LDN–193189 both in vivo and in vitro. Unexpectedly, LDN–193189 administration could dramatically suppress Smad9 level and elevate egg production (7.08 eggs / bird, P< 0.05) of animals, and the estradiol (E2) and luteinizing hormone receptor (LHR) level were significantly increased (P< 0.05), but the progesterone (P4) and follicle stimulating hormone receptor (FSHR) mRNA remain unchanged. Surprisingly, Smad9 knockdown notably attenuated (P< 0.05) in E2, P4, FSHR and LHR level in goose granulosa cells (gGCs). Further chromatin immunoprecipitation (ChIP) assay of gGCs revealed that Smad9, served as a sensor of balance, bound to the LHR promoter regulating its transcription. These findings demonstrated that Smad9 is differentially expressed in goose follicles, and acts as a key player in controlling goose follicular selection.SUMMARY STATEMENTTo study the hierarchical development mechanism of avian follicle, new strategies can be found to improve the egg production of low-yielding poultry, such as geese.